Summary In eukaryotic cells, the process of macroautophagy is the principal catabolic pathway for the degradation of long-lived proteins and its impairment has been linked to a number of proteostatic disorders. Although it is known that macroautophagy can selectively target specific proteins and organelles for lysosomic degradation, the molecular mechanism of this selectivity remains incompletely understood. Here, we are proposing to develop novel proteome-wide approaches to investigate the mechanism of selectivity in macroautophagy. By providing global maps of autophagic flux, we will identify subsets of proteins that rely on macroautophagy for their constitutive turnover and determine the molecular receptors required for their selective degradation. Additionally, we will globally characterize the ability of macroautophagy to selectively target proteins with age-induced damage and clear pathogenic protein aggregates that accumulate during the course of prion diseases. Together, the proposed experiments will provide insights into the mechanism of cargo selection by the autophagy pathway and establish generally applicable proteomic methodologies for quantifying autophagic flux in cultured cells. Furthermore, our studies will provide insights into the role of autophagy in mitigating the proteostatic disruptions that occur during the course of prion diseases, and may identify novel therapeutic approaches targeting these disorders.